Process for producing oxytetra-cycline antibiotic



United States Patent() 3,408,258 PROCESS FOR PRODUCING OXYTETRA- CYCLINE ANTIBIOTIC Arpad Grein, Graziana Canevazzi, and Riccardo Barchielli, Milan, Italy, assignors to Societa Farmacentici Italia, Milan, Italy, a corporation of Italy No Drawing. Filed Feb. 25, 1965, Ser. No. 435,336 Claims priority, application Italy, Feb. 27, 1964, 4,243/ 64 4 Claims. (Cl. 19580) ABSTRACT OF THE DISCLOSURE The present invention relates to a fermentative process for obtaining the antibiotic, oxytetracycline, by means of a new species of St'reptom'yces, named by us Streptomyces henetus or Streptomyces F1. 1782, and which has been deposited at the Institute of Microbiology of the- Rutgers University, U.S.A., where it received the index I.M. 3872. The process of the present invention results in obtaining oxytetracycline in very high yields. These yields are shown to be as high as 11,000 per cc.

described in the literature (S. A. Waksman: The Actinomycetes, vol. III, 1962, p. 329).

We have found that by the process of our present invention, oxytetracycline may be obtained with high yields.

This new microorganism has been isolated from a soil sample taken in the neighborhood of Ramera (Italy) and shows the following morphological, macroscopic, microscopic and biochemical properties:

Microscopic aspect The vegetative myceliumon culture media of potatoagar and Czapek-agar consists of hyphae 0.8-0.9;t thick, of intermediate length, branched and forming aerial mycelium consisting of hyphae from 1.2 to 1.6 thick and not very long. These hyphae abundantly ramify and form straight conidiophores bearing spiralled hyphae then transforming into spores.

The conidia have a smooth surface, are prevalently oval: 1.2 to 1.6, by 1.4 to 1.7a and are disposed first in chains and then free; each chain hearing more than ten spores. The hyphae transforming into spores are attached to conidiophores alternatively or oppositely. The spirals are closed, sometimes forming clews.

Macroscopic aspect In Table I there are listed the cultural properties observed on the media therein indicated, in which the microorganism is grown at 28 C., observations being made at the 3rd 8th, 16th and 21st day after inoculation.

Biochemical properties The microorganism reduces nitrates to nitrites, hydrolyzes starch and gelatin, does not produce hydrogen sulphide, does not coagulate or peptonize milk, and does not produce melanin and digest tyrosine. It produces acids from d-mannose, d-arabinose, glucose, mesoinosite, dfructose, adonitol, maltose and ramnose but not from d-xylose, sorbitol, or saccharose. The microorganism does not form sclerotia and does not grow at C.

TABLE I.CULTURAL PROPERTIES OF STREPTOMYCES RI. 1782 1 S. A. Walrsman: The Actinomycetes, vol. II, 1961, pp. 328-334. 2 Pridham T. G. et al.: Antibiotics Annual 1956-1957, pp. 947-953. 3 Baldacci E; Giom. Microbiol., 1961, 9, p. 39

Media Growth Aerial mycelium Vegetative mycelium Soluble Remarks pigments Bonnet-Agar 1 Abundant, with re Dirty white, smooth, scarce From colorless to straw- Absent.

7 lieved wrinkled yellow to light folds. brownish. Czapek-Agar 1 Scarce Dirty white, scarce with straight Colorless do sporophores. bearing spiralled hyphae transforming into spores. Asparagine-glucose-Agar Moderate Whitish, scanty do do Glycerin-glycine-Agar Dirty white, scanty. do do Emerson-Agar 1 Abundant with Moderate from dirty white to light From straw-yellow to do relieved folds. brownish grey. light brown. Pridham Stareh-Salts-Agar A. Scarce Mediocre of dirty white color straight Colorless do conidiophores bearing spiralled hyr phae transforming into spores. Potato-A gar 4 Abundant From dirty w bite to light brown grey. Frlonill stbraw-yellow to .do ig t rown. Oats-Agar Mod r Scarce, whitish Frorfi coloaless to do ye OWlS Asparagine-glycerin-Agar 1 do d Colorless, yellowish do Starch peptone-Agar 1 do.. do Slightly light brown do Positive hydrolysis. Tyrosine-Agar 5 do Scarce, dirty wh1te Colorless (ln lMIelanin-Agar Scarce.. Very scarce, dirty white do do Gelatin 1 Moderate..- Absent Straw-yellow do Peptone-broth IKNOa do..- -do Frorln honey to yellowish -do co or. Milkdo do"... Straw-yellow do 4 200 g. of peeled potatoes are boiled in water and then filtered through gauze. The filtrate is mixed with 20 g. of glucose and 20 g. of agar. The volume is brought to 1000 cc. and sterilized at C. for 20 minutes.

5 Gordon R. E. et aL: J. Bact., 1955, 69, p. 147.

TABLE II.-COMPLARISON OF sTaEr'roMY oEs F.I. eaiN'D SP ECIES IRODUCTIVE OF OXYTETRACYCLINE S. l t nsis S. armillatus S. vendargus- S. qzlvus Streptoggces F1. S rrmorus p a e vendamemis Sporophorcs l Spiralled Section 8.... Spiralled Section S Spiralled Section S Spiralled Section S StziagghltcSeo- 1715135233 Spores Oval with smooth Cylindrical 0.6-0.7 by Ovoidai 0.7-0.9 by 0.8- Not described Not described. Not described.

surface 1.2-1.6 by 0.8-1.4;1. 1.2a.

1.4-1.7p. V etative m From strawellow to From cream-colored to Strong apricotpr From colorless to a From pale Brown-black.

c elium. y ochre. Revirse side reddish-brown to ochrish-reddish c1 nyellowish gray. yellow to of pale yellow color. orange. narnon color. Apricotbrown. colored reverse side. Aerial mycelium Dirty white, some- From whitish to From whitish to olive Very scarce times up to greystrong (mouse)-gray or mouse-gray to p v I brown. to black. black. I Soluble pigments... Absent Yellowish, some- From yellowish to Sometimes present, Absent Not descrlbed.

- times also brown. yellow-green to brown. gom pale pink to )V v I rown.

-Do. Starch i i D Do. Do. Do Do.

1 According to the Pridham et a1. classification, Appl. Microbiol., 1958, 6, p. 52. Legend in Table II.: Positive reaction; Negative reaction; =1: Very weak reaction.

Identification of the microorganism The description of the microorganism under examination relates to the genus Streptomyces Waksman and Henrici -(Bergeys Manual of Determinative Bacteriolog 7th ed., 1957, pp. 744745). From examination of the species it may be concluded: In the classification system of Pridham et al. (Appl. Microbiol., 1958, 6, p. 52) the microorganism belongs to the section Spira series White; in the classification system of Baldacci (Giorn. Microbiol., 1958, 6, p. 10), the microorganism belongs to the series Albidofiavus; and in the classification systern of Waksman (The Actinomycetes, vol. H, 1961, p. 123) the microorganism belongs to the series Flavus.

A comparison between the properties of the micro organism Streptomyces F1. 1782 and those of species belonging to the above systematic groups (Taxa) demonstrates that Streptomyces RI. 1782 has quite different properties. Besides the differences listed in Table II, Streptomyces F.I. 1782 has a clearly different morphology from Streptomyces rimosus: The hyphae of the former transforming into spores are alternatively or oppositely attached along straight sporophores; while in the latter the hyphae forming spores are attached to short sporophores in a more or less compact fascicled system. Hereinbelow follows a list of the differences from species belonging to the cited Taxa group which do not produce tetracyclines.

Streptomyces RI. 1782 differs from the species S.

flavus, because generally the latter does not produce spirals, in the color of the vegetative and aerial mycelium, and because it does not reduce nitrates (Waksman S.A.: The Actinomycetes, vol. II, 1961, p. 210); from the species S. flavovirens because the latter generally does not produce spirals, forms soluble pigments, and coagulates or peptonizes milk (Waksman, The Actinomycetes, vol. II, 1961, p. 210); from the species S. flavagrz'seus because the latter does not produce spirals and forms a mousegray colored aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 209); from the species S. chrysomallus because the latter does not form spirals and produces soluble pigments (Waksman, The Actinomycetes, vol. II, 1961, p. 193); from the species S. celluloflavus because the latter produces soluble pigments and presents a different color of the aerial mycelium (Waksman, The Actinomycetes, .vol. II, 1961, p. 191);-from the species S. viridans because the latter produces soluble pigments and presents a different color of the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 286); from the species S. 'albidoflavus beca'use'the latter produces soluble pigments and in the color of the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 168); from the species S. citreus because the latter shows a different color of the vegetative and aerial mycelium (Waksman, The Actinomycetes, vol. II, p. 196); from the species'S. parvus because the latter produces soluble pigments and shows a different color of the vegetative and the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 255) from the species S. griseoflavus because the latter shows a different color of the vegetative and the aerial mycelium, furthermore ecause it produces soluble pigments (Waksman, The Actinomycetes, vol. II, 1961, p. 222); from the species S. flavochromogenes because the latter produces soluble pigments and shows a different color of the vegetative and the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 209); from the species S. alboflavus because the latter does not form spirals and because it shows a different color of the vegetative mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 169); from the species S. hygroscopicus because the latter produces soluble pigments and shows a different color of the vegetative mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 230);'from the species S. fimicarius because the latter produces soluble pigments and shows a different color of the vegetative and the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 208); from'the species S. abikoensum because the latter is melanin-positive, does not produce spirals and produces soluble'pi'gments (Waksman, The Actinomycetes, vol. II, 1961, p. from the species S. alboniger because the latter does not produce spirals and shows a different color of the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 170); from the species S. lzachijoenis because the latter does not produce spirals and shows a different color of the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1 961, p. 226); from the species S. eurocidicus because the latter does not produce spirals and because it produces soluble pigments (Waksman, The Actinomycetes, vol. II, 1961, p. 205); from the: species S.

- mediocidicus because the latter does not produce spirals,

is melanin-positive and does not reduce nitrates (Waksman, The Actinomycetes, vol. II, 1961, 242); from the species S.- albidus, v. invertens because the latter belongs to the species S. griseus (Waksman, The Actinomycetes, vol. 11,1961, p. 169) from the species S. albus because the latter produces a snow-white aerial myCelium peptonizes milk and shows different sporophores (Waksman, The Actinomycetes, vol. II, 1961, p. 172); from the speciesS. cacaoi because the latter produces longand open spirals, forms an aerial mycelium which is colored from light-gray to mouse-gray and reduces nitrates weakly (Waksman, The Actinomycetes, vol. II, 1961, p. 183); from the species S. eryrhreus because the latter produces a cream to purple-red aerial mycelium and wineecolored soluble pigments (Waksman, The Actinomycetes, vol. II, 1961, p. 204) from the species S. galtieri because. the latter produces a cream-colored aerial mycelium, a brown soluble pigment and liquefies gelatin weakly (Waksman, The Actinomycetes,vol. II, 1961, p. 215); from the species S. longisporoflavus because the latter produces long, open spirals, an aerial mycelium which is colored from white to lemon, coagulates and peptonizes milk, and hydrolyzes starch weakly (Waksman, The Actinomycetes, vol. II, 1961, p. 37). I 7

It follows therefore that Streptomyces El. 1782 should be considered a species different from those known up to the present and therefore a new species which we have named Streptomyces henetzts.

The microorganism Streptomyces F1. 1782 may be stored by successive cultivation on a solid medium (potato-agar, Sabouraud agar) or by lyophilization, milk being the suspending medium.

The production of the antibiotic Oxytetracycline according to the invention may be performed by cultivating Streptomyces F.I. 1782 in a liquid nutrient medium under aerobic conditions. A temperature of from 24 to 37 C., preferably 28 C., and a period of from 72 to 144 hours have been found satisfactory. The pH may vary from 6.1-6.5 to 8 according to the fermentative media employed.

The cultural medium should consist of a carbon source, a nitrogen source, and salts. The carbon source may be, for example, saccharose, glucose, dextrin, starch, soyabean meal, peanut meal, cotton-seed meal, corn steep liquor, distillers solubles, soyabean oil, or lard oil. The nitrogen source, besides the above complex substances containing nitrogen, may be meat extract, peptone, casein,

hydrolysate of casein and ammonium salts, such as ammonium sulphate or diammonium phosphate. The mineral salts useful for the production of the antibiotic vary according to the medium employed. Calcium carbonate is normally present, and sodium, potassium, manganese, iron, copper, cobalt or zinc chlorides, sulphates or phosphates may be added. The fermentation may be carried out in Erlenmeyer flasks and in laboratory or industrial fermenters of various capacity.

The quantity of antibiotic present in the broth may be qualitatively assessed by paper chromatography in comparison with a standard sample of oxytetracycline and quantitativelychecked by spectrophotometric or biological methods.

The yields obtainable by the process of the invention are as high as 11,000 per cc.

When the fermentation is over, the pH of the culture broth is adjusted to 2-2.5, the mycelium is separated by filtration or centrifugation, and the Oxytetracycline may be recovered from the liquid phase and purified. Useful methods of purification are: extraction with an organic solvent, for example butanol, pentanol, hexanol, amyl alcohol, methylethylketone, methyl isobutylketone or butyl acetate; adsorption on a solid substance, for example activated charcoal, and subsequent elution with water or an organic solvent; precipitation of the antibiotic in the form of one of its derivatives or complex salts bymeans, for example, of an amine, a quaternary ammonium compound in the presence of a metal ion, ammonia or sodium carbonate, urea, N,N -malonylurea, sulphonic derivatives or sulphates. Oxytetracycline thus obtained may be then purified by recrystallization or transformed into its salts with a mineral or organic acid, such as hydrochloric, sulphuric, citric or tartaric acid.

The present invention includes a method of extraction and purification of the oxytetracycline produced, which comprises extracting the acidified culture broth, filtering the product and treating the resulting filtrate with oxalic acid, filtering, making alkali, extracting with a mixture of tricresol and carbon tetrachloride, separating the organic phase, treating it with aqueous hydrochloric acid and acetone and separating the aqueous phase. In a preferred embodiment, the culture broth is acidified with a mineral acid, for'exarnple hydrochloric, sulphuric or phosphoric acid, to pH 2-2.5, is filtered and the filtrate obtained is first treated with oxalic acid and then filtered to remove precipitated calcium oxalate; the filtrate is extracted with a mixture of tricresol and carbon tetrachloride adjusting the pH to 8-8.5 by addition of alkali. The tricresol em ployed is that generally known and employed for industrial uses, having a specific gravity 1.0301.038, consisting of a mixture of the three isomeric cresols. It is preferable to employ a mixture of tricresol and carbontetrachloride in a ratio 2:1 to 1:2 by volume. The phases thus obtained are separated by centrifuging and to the separated organic phase, which contains the antibiotic, a mixture of equal volumes of acetone and aqueous hydrochloric acid, preferably 1 N hydrochloric acid, is added. From the aqueous phase thus obtained and adjusted to pH 7, by addition of an alkaline material such as an alkali metal hydroxide or carbonate, the antibiotic Oxytetracycline precipitates and is isolated and purified by recrystallization or transformed into a salt with a mineral or organic acid.

The followng examples illustrate the invention, without intent to limit it.

EXAMPLE 1 Into each of two 300 cc. Erlenmeyer flasks, 60 cc. of the following medium is poured:

Percent Dextrin 3 Calcium carbonate 0.4 Corn steep liquor 0.3 Casein a 0.5 Ammonium sulphate 0.1 Potassium hydrogen phosphate (K HPO 0.01

Tap water, to 1000 cc.

and sterilized by heating in an autoclave to C. for 20 minutes. The pH after sterilization is 6.8-7.0. Each flask is inoculated with 1.5 cc. of a suspension of spores obtained by scraping off, into 5 cc. of distilled sterile Water, the surface of a 20 days old slant of a culture of Streptomyces RI. 1782 grown on the following solid medium: 200 g. of peeled potatoes are boiled for about 20 minutes in 500 cc. of water. The volume is brought to its original value and filtered through gauze. 2% of glucose and 2% of agar are added. The volume is brought to 1000 cc. and sterilized in an autoclave at 120 C. for 20 minutes. The pH of the medium is 6.8-7.0. The flasks are incubated at 28 C. for 24 hours on a rotary shaker with a stroke of 30 mm. at 220 rpm. 1 cc. of a culture thus grown is used to inoculate Erlenmeyer flasks of 300 cc. with 60 cc. of the following productive medium:

Tap water, to 1000 cc.

The sterilization is carried out at 120 C. for 20 minutes. The pH after sterilization is 6770. It is incubated at 8 C. under the conditions described above for the vegetative medium. 4000 per cc. of Oxytetracycline is produced after 96 hours of fermentation.

EXAMPLE 2 The operation is as in Example 1, with the difference that the culture to be inoculated is 10 days old and is grown on the following medium:

Percent Peptone 0.8 Glucose 4 Difco agar 2 Tap water, to 1000 cc.

. sterilized in an autoclave at 120 C. for about 1 minute. The pH is 6.8-7.0. In addition the productive phase differs in-the composition of the medium being as follows:

7 Percent Starch 8 Corn steep liquor t 2.5

7 Calcium carbonate -Q. i 1 Ammonium sulphate v 1 Manganese sulphate 0015 Cobalt nitrate 0.00075 Cotton seed meal 0.5

Lard oil Tap water, to 1000 cc.

sterilized in an autoclave at 120 C; for 20 minutes. The pH of the medium after sterilization is 6.5 -6.8. It is incubated at 28 C. under the same shaking conditionsas in Example 1. 8500 per cc. of oxytetracycline is obtained after 144 hours of fermentation.

EXAMPLE 3 Operation is as in Example 2 with the difference that the culture is 20 days old and the vegetative phase is carried out on the following liquid medium:

' Percent Corn steep liquor 3.5 Calcium carbonate 1.8

Saccharose 0.5

Distilled water, to 1000 cc.

sterilized in an autoclave at 120 C. for 20 minutes. The pH after sterilization is 6.2-6.3. Incubation is for 26 hours at 28 C. on a rotary shaker with a stroke of 60 mm. at 220 r.p.m. The productive phase is carried out in 300 cc.

flasks with 35 cc. of the following productive medium.

Percent Starch 8 Corn steep liquor 2.6 Calcium carbonate '1 Ammonium sulphate l Manganese sulphate 0.014 Cobalt chloride 0.00078 Cotton seed meal 0.43

1 cc. of lard oil for each flask containing 35 cc.

of the medium.

The pH after sterilization is 6.5-6.7. The medium is sterilized in an autoclave at 120 C. for 20 minutes. The quantity of the vegetative medium to be inoculated into the productive medium is 4.3% of the latter. The incubation of the productive phase is carried out for 24 hours at 28 C. and then at 24 C. for 120 hours. 7600 per cc. of oxytetracycline are obtained.

EXAMPLE 4 Operation is as in Example 2 with the difference that operation is always at 24 C. The production obtained in 96 hours of fermentation is 6400 per cc. of oxytetracycline.

EXAMPLE and a temperature checking equipment. Growth is carried out at 28 C. with an aeration rate of 3 liters per minute, and with shaking at a rate of 400 r.p.m. After 24hours,

150 cc. of the culture broth thus grown are inoculated into 3 liters of the following productive medium:

Percent Fatless'soyabean" meal 3 Distillers solubles 0.5

Glucose I 2 Sodium chloride Tap wa'ter,'to 1000 cc. sterilized by heating in an autoclave at C. for 20 minutes. The pH after sterilization is 6.5. The above medium is contained ma 5 liter neutral glass fermentor, having the above-described proper-ties. During the fermentation, carried out at a stirring rate of 500 r.p.m. and with an aeration rate of 3 liters per minute, foaming is checked by adding small amounts ofv silicone antifoaming agent. The highest production of oxytetracyclineobtain ed in 88 hours of fermentation corresponds to 58007 per cc.

EXAMPLE 6 (Purification) .The pH of 333 liters of a broth culture having an antibiotic concentration of 4500 per cc., obtained asdescribed in any of the preceding examples, is adjusted to 2 by adding dilute 25% aqueous sulphuric acid. The culture broth is filtered with 1 kg. of infusorial earth and the filtration cake is washed with acidulated water (pH 2- sulphuric acid) so that the volume of the filtrate reaches 45 liters. 2.2 kg. of oxalicacid are added and the mixture is stirred for 30 minutes, filtered and the filter washed with water so that the volume of the filtrate reaches 50 liters. 50 liters of this broth filtered and decalcified, containing g. of oxytetracycline and poured into a steel vessel, are shaken with a mixture of 2.5 liters of tricresol and 2.5 liters of carbon tetrachloride, the pH of the solution being adjusted to 8.3 with an aqueous solution of sodium hydroxide. The two layers making up the mixture are separated by centrifuging. The aqueous layer is extracted once again with a mixture of 1 liter of tricresol and 1 liter-of carbon tetrachloride. The lower heavy organic layers are combined and poured into a mixture of 5 liters of acetone and 5 liters of aqueous 1 N hydrochloric acid and thoroughly shaken. The two'layers are. separated by centrifuging. The heavy layer is extracted twice again with 3 liters-and 2 liters respectively of 1 N aqueousrhydrochloric acid. The pH of the combined aqueous layers (10 liters) is adjusted to 7 by adding with stirring a dilute (20%) aqueous sodium hydroxide. A precipitate separates, which is recovered by filtration, washed with diethyl ether and dried in vacuo at 40 C. The residue is ground, washed again with 'etherand dried at 40 C. 150 g. of crude oxytetracycline are obtained.

150 g. of this product are suspended in 1.5 liters of methanol and dissolved byaddition of 200 cc. of 13% hydrochloric acid in methanol. To the solution 14 g. of decolorizing carbon are added, it is stirred-for 30 minutes, filtered on infusorial earth and washed with methanol. The filtrate is concentrated in vacuo to a volume of about 400cc and 30 cc. of 13% aqueous hydrochloric acid in methanol are added. On rubbing, a product-crystallizes and the-mixture is allowed to stand overnight'at-room temperature, filtered, washed first with 50 cc. of methanol and then with diethyl ether, and dried at 40 .C. in vacuo. 95 g. of oxytetracycline hydrochloride are obtained.

} EXAMPLE 7 1 Operation is as in Example 3, with thedifference that the vegetative phase and productive phase are carried out at 27- C. and that the productivemedium amount contained in. each flask is 50 cc. A production of 11,0007 per cc. of oxytetracycline is'obtain'ed after'6-7 days of fermentation.'-

1. A process for preparing oxytetracycline, which comprises cultivating the new' microorganism Streptomyces henetus Rutgers University Institute of Microbiology I.M.

3872 under aerobic conditions in a nutrient medium containing a source of carbon, nitrogen and mineral salts, at a temperature of from 24 to 37 C., over a period of from 72 to 144 hours, at pH 6.1 to 8, and separating the oxytetracycline thus formed from the fermentation broth and purifying the separated oxytetracycline.

2. A process for preparing oxytetracycline, which comprises cultivating the new microorganism Streptomyces lzenetus Rutgers University Institute of Microbiology I.M. 3872 under aerobic conditions in a nutrient medium containing a source of carbon, nitrogen and mineral salts, at a temperature of from 24 to 37 C., over a period of from 72 to 144 hours, at pH 6.1 to 8, acidifying the culture broth containing oxytetracycline to pH 2-2.5 with an inorganic acid, filtering and treating the filtrate with oxalic acid and filtering to remove calcium oxalate precipitated and making alkaline the filtrate obtained to pH 8-8.5 by adding an inorganic base and extracting with a mixture of tricresol and carbon tetrachloride, and transferring the oxytetracycline from the organic phase into an aqueous phase by a mixture of aqueous hydrochloric acid and acetone, and precipitating the oxytetracycline from the aqueous phase thus obtained by previous neutralization to pH 7, and purifying the oxytetracycline.

3. A process for preparing oxytetracycline, which comprises cultivating the new microoganism Streptomyces henetus Rutgers University Institute of Microbiology I.M. 3872 under aerobic conditions in a nutrient medium containing a source of carbon, nitrogen and mineral salts, at a temperature of from 24 to 37 C., over a period of from 72 to 144 hours, at pH 6.1 to 8, acidifying the cuture broth containing oxytetracycline to pH 2-2.5 with an inorganic acid, filtering and treating the filtrate with oxalic acid and filtering to remove calcium oxalate pre cipitated and making alkaline the filtrate obtained to pH 8-8.5 by adding an inorganic base and extracting with a mixture of tricresol and carbon tetrachloride in a ratio of 2:1 to 1:2 by volume, and transferring the oxytetracycline from the organic phase into an aqueous phase by 10 a mixture of aqueous hydrochloric acid and acetone, and precipitating the oxytetracycline from the aqueous phase thus obtained by previous neutralization to pH 7, and purifying the oxytetracycline.

4. A process for preparing oxytetracycline, which comprises cultivating the new microorganism Streptomyces henetus Rutgers University Institute of Microbiology I.M. 3872 under aerobic conditions in a nutrient medium containing a source of carbon, nitrogen and mineral salts, at a temperature of from 24 to 37 C., over a period of from 72 to 144 hours, at pH 6.1 to 8, acidifying the culture broth containing oxytetracycline to pH 2-2.5 with an inorganic acid, filtering and treating the filtrate with oxalic acid and filtering to remove calcium oxalate precipitated and making alkaline the filtrate obtained to pH 8-8.5 by adding an inorganic base and extracting with a mixture of tricresol and carbon tetrachloride, and transferring the oxytetracycline from the organic phase into an aqueous phase by a mixture of aqueous 1 N hydrochloric acid and acetone in an equal ratio by-volume, and precipitating the oxytetracycline from the aqueous phase thus obtained by previous neutralization to pH 7, and purifying the oxytetracycline.

References Cited UNITED STATES PATENTS 7/1950 Sobin et al. -80 X FOREIGN PATENTS 112,256 11/1962 Pakistan.

OTHER REFERENCES MAURICE W. GREENSTEIN, Primary Examiner. 

